Blurred lines: Multiple freshwater and marine algal toxins...

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Blurred lines: Multiple freshwater and marine algal toxins at the land-sea interface of San Francisco Bay, California Melissa B. Peacock a,b,c, *, Corinne M. Gibble b,d , David B. Senn d , James E. Cloern e , Raphael M. Kudela b a Northwest Indian College, 2522 Kwina Rd, Bellingham, WA, 98226, USA b Ocean Sciences Department, 1156 High Street, University of California, Santa Cruz, CA 95064, USA c San Francisco Estuary Institute, 4911 Central Avenue, Richmond, CA 94804, USA d California Department of Fish and Wildlife, Ofce of Spill Prevention and Response, Marine Wildlife Veterinary Care and Research Center, 151 McAllister Way, Santa Cruz, CA 95060, USA e United States Geological Survey MS496, 345 Middleeld Rd, Menlo Park, CA 94025, USA A R T I C L E I N F O Article history: Received 9 September 2017 Received in revised form 10 February 2018 Accepted 10 February 2018 Available online xxx Keywords: Amnesic shellsh poisoning Paralytic shellsh poisoning Diarrhetic shellsh poisoning Microcystin toxins Shellsh Chronic toxin exposure A B S T R A C T San Francisco Bay (SFB) is a eutrophic estuary that harbors both freshwater and marine toxigenic organisms that are responsible for harmful algal blooms. While there are few commercial shery harvests within SFB, recreational and subsistence harvesting for shellsh is common. Coastal shellsh are monitored for domoic acid and paralytic shellsh toxins (PSTs), but within SFB there is no routine monitoring for either toxin. Dinophysis shellsh toxins (DSTs) and freshwater microcystins are also present within SFB, but not routinely monitored. Acute exposure to any of these toxin groups has severe consequences for marine organisms and humans, but chronic exposure to sub-lethal doses, or synergistic effects from multiple toxins, are poorly understood and rarely addressed. This study documents the occurrence of domoic acid and microcystins in SFB from 2011 to 2016, and identies domoic acid, microcystins, DSTs, and PSTs in marine mussels within SFB in 2012, 2014, and 2015. At least one toxin was detected in 99% of mussel samples, and all four toxin suites were identied in 37% of mussels. The presence of these toxins in marine mussels indicates that wildlife and humans who consume them are exposed to toxins at both sub-lethal and acute levels. As such, there are potential deleterious impacts for marine organisms and humans and these effects are unlikely to be documented. These results demonstrate the need for regular monitoring of marine and freshwater toxins in SFB, and suggest that co-occurrence of multiple toxins is a potential threat in other ecosystems where freshwater and seawater mix. © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Harmful Algal Blooms (HABs) cause health problems for humans and wildlife, as well as persistent ecosystem damage. Both frequency and intensity of HABs are increasing (Anderson et al., 2002; Anderson, 2009; Hallegraeff, 1993; Wells et al., 2015). Impacts from human induced-nutrient inputs from agriculture and urban runoff, global warming, and drought events increase the likelihood that HABs and their impacts will continue to expand (Lehman et al., 2017; Paerl and Paul, 2012). This expansion is occurring in both freshwater and marine environments, but to date, there have been few studies examining the co-occurrence of freshwater and marine toxins in estuaries (Lopes and Vasconcelos, 2011; Miller et al., 2010; Rita et al., 2014; Vasconcelos, 1995), in part because management and monitoring of HABs are often partitioned as either freshwater or marine (Gibble et al., 2016; Preece et al., 2017). Both marine and freshwater toxigenic HAB species have been documented in San Francisco Bay (SFB) for the last three decades (Cloern and Dufford, 2005; Kudela et al., 2008; Lehman et al., 2010; Nejad et al., 2017). Marine HABs likely enter SFB from the Pacic Ocean (Horner et al., 1997), while freshwater HABs are dominant upstream and are likely transported into the estuary during high- ow events (Lehman et al., 2005), though SFB and the surrounding environs, such as the South Bay salt ponds, also contain resident populations of HAB organisms (Thébault et al., 2008). In part because of legacy anthropogenic contamination, there are few commercial * Corresponding author at: Northwest Indian College, Bellingham, WA, 98226, USA. E-mail address: [email protected] (M.B. Peacock). https://doi.org/10.1016/j.hal.2018.02.005 1568-9883/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Harmful Algae 73 (2018) 138147 Contents lists available at ScienceDirect Harmful Algae journal home page : www.elsevier.com/locat e/hal

Transcript of Blurred lines: Multiple freshwater and marine algal toxins...

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Harmful Algae 73 (2018) 138–147

Blurred lines: Multiple freshwater and marine algal toxinsat the land-sea interface of San Francisco Bay, California

Melissa B. Peacocka,b,c,*, Corinne M. Gibbleb,d, David B. Sennd, James E. Cloerne,Raphael M. Kudelab

aNorthwest Indian College, 2522 Kwina Rd, Bellingham, WA, 98226, USAbOcean Sciences Department, 1156 High Street, University of California, Santa Cruz, CA 95064, USAc San Francisco Estuary Institute, 4911 Central Avenue, Richmond, CA 94804, USAdCalifornia Department of Fish and Wildlife, Office of Spill Prevention and Response, Marine Wildlife Veterinary Care and Research Center, 151 McAllisterWay, Santa Cruz, CA 95060, USAeUnited States Geological Survey MS496, 345 Middlefield Rd, Menlo Park, CA 94025, USA

A R T I C L E I N F O

Article history:Received 9 September 2017Received in revised form 10 February 2018Accepted 10 February 2018Available online xxx

Keywords:Amnesic shellfish poisoningParalytic shellfish poisoningDiarrhetic shellfish poisoningMicrocystin toxinsShellfishChronic toxin exposure

A B S T R A C T

San Francisco Bay (SFB) is a eutrophic estuary that harbors both freshwater and marine toxigenic organismsthat are responsible for harmful algal blooms. While there are few commercial fishery harvests within SFB,recreational and subsistence harvesting for shellfish is common. Coastal shellfish are monitored for domoicacid and paralytic shellfish toxins (PSTs), but within SFB there is no routine monitoring for either toxin.Dinophysis shellfish toxins (DSTs) and freshwater microcystins are also present within SFB, but notroutinely monitored. Acute exposure to any of these toxin groups has severe consequences for marineorganisms and humans, but chronic exposure to sub-lethal doses, or synergistic effects from multipletoxins, are poorly understood and rarely addressed. This study documents the occurrence of domoic acidand microcystins in SFB from 2011 to 2016, and identifies domoic acid, microcystins, DSTs, and PSTs inmarine mussels within SFB in 2012, 2014, and 2015. At least one toxin was detected in 99% of musselsamples, and all four toxin suites were identified in 37% of mussels. The presence of these toxins in marinemussels indicates that wildlife and humans who consume them are exposed to toxins at both sub-lethaland acute levels. As such, there are potential deleterious impacts for marine organisms and humans andthese effects are unlikely to be documented. These results demonstrate the need for regular monitoring ofmarine and freshwater toxins in SFB, and suggest that co-occurrence of multiple toxins is a potential threatin other ecosystems where freshwater and seawater mix.© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents lists available at ScienceDirect

Harmful Algae

journal home page : www.elsevier .com/ locat e/hal

1. Introduction

Harmful Algal Blooms (HABs) cause health problems forhumans and wildlife, as well as persistent ecosystem damage.Both frequency and intensity of HABs are increasing (Andersonet al., 2002; Anderson, 2009; Hallegraeff, 1993; Wells et al., 2015).Impacts from human induced-nutrient inputs from agriculture andurban runoff, global warming, and drought events increase thelikelihood that HABs and their impacts will continue to expand(Lehman et al., 2017; Paerl and Paul, 2012). This expansion isoccurring in both freshwater and marine environments, but to

* Corresponding author at: Northwest Indian College, Bellingham, WA, 98226,USA.

E-mail address: [email protected] (M.B. Peacock).

https://doi.org/10.1016/j.hal.2018.02.0051568-9883/© 2018 The Authors. Published by Elsevier B.V. This is an open access article un

date, there have been few studies examining the co-occurrence offreshwater and marine toxins in estuaries (Lopes and Vasconcelos,2011; Miller et al., 2010; Rita et al., 2014; Vasconcelos, 1995), inpart because management and monitoring of HABs are oftenpartitioned as either freshwater or marine (Gibble et al., 2016;Preece et al., 2017).

Both marine and freshwater toxigenic HAB species have beendocumented in San Francisco Bay (SFB) for the last three decades(Cloern and Dufford, 2005; Kudela et al., 2008; Lehman et al., 2010;Nejad et al., 2017). Marine HABs likely enter SFB from the PacificOcean (Horner et al., 1997), while freshwater HABs are dominantupstream and are likely transported into the estuary during high-flow events (Lehman et al., 2005), though SFB and the surroundingenvirons, such as the South Bay salt ponds, also contain residentpopulations of HAB organisms (Thébaultet al., 2008). Inpart becauseof legacy anthropogenic contamination, there are few commercial

der the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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fisheries within SFB, but recreational and subsistence shellfishharvesting is common (Davis et al., 2002; Gibble et al., 2016). Acuteexposures to toxigenic algae can have severe and deadly impacts onhigher trophic levels (Lefebvreetal., 2012; Shumwayet al.,2003; VanDolah et al., 2002). Sub-lethal chronic doses may also produceserious health compromises in humans (Capper et al., 2013; Ferrisset al., 2017), but chronic exposure to algal toxins has been poorlydocumented, and is an emerging concern (Brooks et al., 2012; Geret al., 2010, 2009; Preece et al., 2017).

Although multiple HAB species are present in SFB, the toxinsmost commonly documented in the food web are domoic acid,produced by the diatom genus Pseudo-nitzschia, and microcystins,produced by the cyanobacteria genus Microcystis (Gibble et al.,2016; Lehman et al., 2010; McHuron et al., 2013). Human andwildlife acute exposure to domoic acid presents as AmnesicShellfish Poisoning (ASP; Lefebvre et al., 2012), and is regulated bythe California Department of Public Health (CDPH) for commercialand recreational shellfish harvests in coastal waters followingfederal guidelines (Wekell et al., 2004). Microcystins are a family ofstrong hepatotoxins causing liver damage in humans and wildlife(Carmichael et al., 2001), and have been documented in bothestuarine and marine waterways, including SFB (Gibble et al., 2016;Gibble and Kudela, 2014; Lehman et al., 2005; Miller et al., 2010).Microcystins have been detected in marine mussels within SFB(Gibble et al., 2016), further documenting the occurrence offreshwater toxins in the marine environment (Gibble et al., 2016;Gibble and Kudela, 2014; Miller et al., 2010; Preece et al., 2017). Todate, current monitoring has been effective at preventing ASP inhumans, though mortality events in marine wildlife are common(McCabe et al., 2016; McKibben et al., 2017). The CDPH does notroutinely monitor for microcystins, though episodic sampling doestake place when blooms are documented.

In addition to microcystins and domoic acid, other algal toxins arelikely to occur in SFB but are not routinely monitored. This includes theparalytic shellfish toxins (PSTs), causative agents for Paralytic ShellfishPoisoning (PSP) in wildlife and humans and primarily associated withthe marine dinoflagellate genus Alexandrium (Horner et al., 1997). Afourth toxin suite evaluated in this study is produced by the marinedinoflagellate genus Dinophysis, comprised of the lipophilic toxinsokadaic acid and derivatives (i.e., dinophysistoxin-1 and -2)collectively referred to as Dinophysis Shellfish Toxins (DSTs). TheDSTs are the causative agent for Diarrhetic Shellfish Poisoning (DSP)in humans (Reguera et al., 2012; Yasumoto et al., 1978). In SFBDinophysis is rare and when present does not dominate thephytoplankton community (Cloern et al.,1985; Cloern and Dufford,2005),butevenat lowabundancesDinophysishasbeenlinkedtoDSPoccurrences (Rundberget et al., 2009). The CDPH routinely monitorscoastal shellfish with an actionable regulatory limit for PSTs (Wekellet al., 2004), but there is currently no routine monitoring forDinophysis or DSTs for either coastal California or SFB.

Here, this study documents co-occurrence of all four toxinsuites in the marine mussel Mytilus californianus during 100-daydeployments in 2012 and 2014, and in the hybridized Bay/Mediterranean marine mussel (M. trossulus and M. galloprovincia-lis) during a 5-month period in 2015. The occurrence of domoicacid and microcystins in both particulate and dissolved phasesthroughout SFB from 2011 to 2016 are also recorded.

2. Methods

2.1. Study area

The SFB is the largest estuary in California, consisting of six sub-embayments defined by their characteristic ranges of water-quality variables (Jassby et al., 1997). Spatial patterns in thisestuary are controlled by the salinity gradient and its seasonal

variability (Cloern et al., 2017). Most freshwater flow into SFBcomes from the Sacramento and San Joaquin Rivers draining 40% ofCalifornia’s landscape. Runoff from the agricultural Central Valley,urban sources, and treated sewage and storm-water contribute tohigh nutrient inputs. As a result, dissolved nutrient concentrationsin SFB equal or exceed those in other estuaries where nutrientenrichment has degraded water and habitat quality (Cloern andJassby, 2012). Thus, the potential for HAB development is high inthis enriched estuary.

This study encompasses a period of severe drought (2012–2016), with record high temperatures and salinity in water year2015 (October 1, 2014 to September 30, 2015; Work et al., 2017),and record low flows (Lehman et al., 2017). These conditionsincreased the intensity and duration of Microcystis blooms in theSFB/Delta system (Lehman et al., 2017). At the same time, unusualconditions along the open coast associated with the Pacific WarmAnomaly (Bond et al., 2015), including a massive Pseudo-nitzschiabloom with correspondingly high levels of domoic acid (McCabeet al., 2016) are expected to drive both physical and biologicalchanges inside SFB through its connectivity with the open coast(Cloern et al., 2017; Raimonet and Cloern, 2017).

2.2. Field samples

2.2.1. Particulate grab (filter) samplesWater samples were collected during USGS water quality

cruises within SFB from November 2011 to June 2016 roughly twicemonthly. Surface water (1–2 m) was filtered under low-vacuumpressure to collect particulate toxins. The same grab sample filterwas used to analyze both domoic acid and microcystins. Ancillarydata from these stations are available from the United StatesGeological Survey (USGS), including phytoplankton microscopysamples (Cloern and Dufford, 2005; Nejad et al., 2017).

2.2.2. Solid Phase Adsorption Toxin Tracking (SPATT) samplersIn addition to analyses of particulate toxins, an indicator of

integrated dissolved toxin along subembayment transects was alsoused. Sample bags of SPATT were constructed and activated with3 g (dry weight) DIAION HP20 (Sorbent Technologies) in 100 mmnitex (Wildlife Supply Company) mesh bags (Kudela, 2011; Laneet al., 2010). The SPATT bags were clamped into plastic embroideryhoops and secured in a container with continuous underway flowfrom 0.5 m using a through-hull pumping system and weredeployed on five transect lines. Fan et al. (2014) demonstrated thatSPATT with HP20 exhibit non-linear effects on adsorption ofokadaic acid and dinophysistoxin-1 in laboratory studies. Given thesalinity gradients in San Francisco Bay, salinity probably intro-duced variability in the SPATT data but it is not possible to correctfor this effect without better characterization of this effect in field-deployed samples for a full range of toxins.

2.2.3. Mussel collectionsM. californianus were collected from Bodega Head, CA (Fig. 1) in

May 2012 and 2014 and held in filtered seawater tanks for onemonth to allow depuration of toxins (Bricelj and Shumway, 1998).Cages with mussels were attached to moorings throughout SFB(June 2012 and 2014), at all stations marked on Fig. 1. Mussels wereharvested for analysis in September each year, after 100 daysdeployment. Mussels were homogenized for analysis (60 mussels,2012; 110 mussels, 2014) and 5–20 g, a function of the sizedistribution of the available mussels, of homogenized tissue wasstored frozen until toxin analysis.

Environmental hybridized M. trossulus and M. galloprovincialiswere collected in SFB monthly from April to September 2015 at fourlocations: Point Isabel, Point Potrero, Berkeley Marina, and AlamedaIsland, with 15 individual mussels collected per site and sampling

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Fig.1. Map of San Francisco Bay. Left panel shows the particulate (filter) and dissolved (SPATT) stations, and the right panel shows the mussel collection or placement stations.Placed mussels are indicated as squares (2012 only) and triangles (2012 and 2014) and natural mussel collection sites (2015) are indicated as circles.

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event. Romberg-Tiburon Center was included from July to Septem-ber 2015 (Fig.1). Salinities at these sites varied between 27 and 32 atcollection time (Cloern and Schraga, 2016). The microcystin data hasbeen published (Gibble et al., 2016). For this study a randomlyselected subset of 3 of the 15 mussels were chosen for toxin analysis,as described below. There was insufficient biomass to test for alltoxins, so sample sizes ranged from n = 34 to n = 81.

2.3. Domoic acid analysis

Particulate grab (filter) samples were analyzed via LiquidChromatography Mass Spectrometry (LC/MS) for domoic acid(Lane et al., 2010). 1.5 mL of the extract was stored (�80 �C) forprocessing of microcystins. Samples were purified on BondElut C18Solid Phase Extraction (SPE) columns (Agilent, USA; Sison-Manguset al., 2016) with an elution volume of 3.0 mL.

For extraction of domoic acid, SPATT were rinsed with Milli-Qwater and processed (Lane et al., 2010) with the followingmodification: bags were cut open and the resin was extractedout of bag without vortex mixing. Following the first 10 mL 50%MeOH extraction, a second extraction with 10 mL of 1 Mammonium acetate in 50% MeOH, and a third extraction with20 mL of 1 M ammonium acetate in 50% MeOH was completed.Extracts were analyzed separately and summed for total toxin,then normalized to the resin dry weight to provide units of mgdomoic acid per kg resin (mg kg�1; Lane et al., 2010).

Mussel samples for domoic acid analysis were prepared asfollows: 1 g of homogenized mussel from each individual wasadded to 10 mL of 50% MeOH and disrupted by ultrasonic probe for30 s with output power of <10 W. Extracts were clarified through a0.22 mm PTFE filter and 3 mL was cleaned using Biotage ISOLUTESAX SPE columns pre-conditioned with 6 mL of 100% MeOH, 3 mLof Milli-Q, and 3 mL of 50% MeOH. The mussel sample was added tothe column, followed by 3 mL of acetonitrile: MilliQ: formic acid in(10:88:2 by volume) to elute the toxin, which was analyzed by LC/MS (Lane et al., 2010). The Method Detection Limit (MDL) was

0.30 mg kg�1 for tissue, 7.50 ng L�1 for particulate samples, and0.87 mg kg�1 for SPATT. Domoic acid is reported as the sum ofdomoic acid and epi-domoic acid, and quantified by peak area andretention time using an external standard curve and NRC-CanadaCertified Reference Material (NRC-CCRM).

2.4. Microcystin toxins analysis

Particulate grab samples were processed using the set aside 1.5 mLof domoic acid preparation (see above) that had been stored at �80 �C,and re-disruption of the filter following Lane et al., 2010, but with afinished extract concentration of 90% acidified MeOH before cleaningusing methods of Mekebri et al., 2009 for saltwater mussels. Briefly, a375 mL aliquot of the previously prepared domoic acid extract (in 10%MeOH) was set aside (labeled here as extract A), and 750 mL of theremaining 10% MeOH extract with the addition of the disrupted filterwas re-disrupted as above and extracted in 6 mL of 100% MeOH.Extract was filtered through a 0.22 mm PTFE filter. 1 mL of 90% MeOHextract was set aside for archival purposes, and extract A was added toproduce 6.125 mL of total extract (combined and labeled here asextract B) for a final concentration of 90% MeOH. Extract B was thenacidified with 6 mL of trifluoroacetic acid (Mekebri et al., 2009). Thisprocess allowed particulate samples to be analyzed for both domoicacid (in 10% MeOH) and microcystins (in 90% acidified MeOH). Beforeanalysis, microcystin samples were then cleaned using BAKER-BONDTM C18 SPE disposable columns and the collected elution wasanalyzed by LC/MS (Gibble et al., 2016). Analysis of extract A and Bseparately by LC/MS indicated that differences were within themethod error for replicates, as were replicates which were not firstextracted in 10% MeOH for domoic acid.

The SPATT bags were rinsed with deionized water andprocessed (Kudela, 2011). Mussel analysis for microcystins followsthe field sample method analysis of Gibble et al. (2016), with theaddition of a 45 min sonicating water bath following probesonication using a Fisher Scientific Sonic Dismembrator Model 100at 8 W power for 30 s.

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Fig. 2. Particulate toxin grab (filter) samples taken during USGS cruises from November 2011 to June 2016. Top panel is domoic acid concentrations, bottom panel ismicrocystin concentrations. Stations can be seen in Fig. 1.

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Mussel samples for microcystin analysis were preparedaccording to Gibble et al., 2016. Grab, SPATT, and mussel sampleswere analyzed by LC/MS (Gibble et al., 2016; Mekebri et al., 2009).The MDL for microcystins was 0.10 mg kg�1 for tissue, 0.10 ng L�1

for particulate samples, and 0.05 mg kg�1 for SPATT. Microcystinsare reported as the sum of four congeners: MC-LR, MC-RR, MC-LA,and MC-YR. This follows the recommended guidelines for theCalifornia Office of Environmental Health Hazard Assessment (Butler et al., 2009).

2.5. PSTs analysis

Mussels were prepared for PST analysis following MaxSignal1

Saxitoxin (PSP) Enzyme-Linked Immunosorbant Assay (ELISA) TestKit Manual instructions (BIOO Scientific, USA). The reporteddetection limit from the manufacturer is 3 mg kg�1 for musseltissue as saxitoxin equivalents, with reported cross-reactivityvarying from 4 to 100% for the various derivatives, and optimizedfor saxitoxin and decarbamoyl saxitoxin.

2.6. DSTs analysis

Mussels were prepared for DST analysis following extractionand hydrolysis by Villar-González and Rodríguez-Velasco (2008).Samples were quantified for DTX-1, DTX-2, and okadaic acid, usingan external standard curve with NRC-CCRM standards. The MDLwas 3.44 mg kg�1 tissue. Concentrations of DSTs are reported as thesum of DTX-1, DTX-2, and okadaic acid.

3. Results

3.1. Particulate toxins in grab samples

Fig. 1 provides an overview of sampling locations, while Fig. 2shows spatial and temporal patterns of toxin variability measuredin particulate grab samples collected in SFB. The minimum,

maximum, mean, median, and number of samples for each stationare provided in Table 1. Eleven percent (n = 420) of grab sampleshad measurable domoic acid, and 51% (n = 405) had detectablemicrocystins. In 3% of samples there was detection of both domoicacid and microcystins. There was no correlation between presenceof toxin and phytoplankton cell counts of toxigenic organisms foreither toxin group.

3.2. Dissolved toxins in Solid Phase Adsorption Toxin Tracking (SPATT)samples

Toxin was detectable in 97% (n = 211) of SPATT in varyingconcentrations for domoic acid, and was present in both themarine and brackish/freshwater segments of SFB. Concentrationswere elevated across all segments in the fall of 2014 through thespring of 2015. Microcystins were detected in 76% of SPATT(n = 210), most often in the freshwater Northern Estuary of SFB, andfrequently in the marine portions. Combined, 100% of SPATT werepositive for either domoic acid or microcystins (Fig. 3, Table 2),with 73% positive SPATT for both toxins.

3.3. Toxins in mussels

In 2012 (n = 11) and 2014 (n = 6) mussel homogenates fromtransplanted mussels obtained from Bodega Head, CA, were testedfor all four toxin suites, including reference samples from BodegaHead. Domoic acid was detected in 100% of mussels for 2012 and2014, including Bodega Head, ranging from 20.5 to 565 mg kg�1.Microcystins were detected in 82% (2012) and 100% (2014) ofmussel homogenates, but not in the reference mussels fromBodega Head. Concentrations ranged from <MDL to 18.9 mg kg�1.PSTs were detected in 45% (2012) and 83% (2014) of homogenates,as well as in the reference mussels in 2014 (25.7 mg kg�1), rangingfrom <MDL to 34.3 mg kg�1. DSTs were found in 91% (2012) and100% (2014) of homogenates, including reference mussels in 2012and 2014. Concentrations in SFB ranged from <MDL to

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Table 1Particulate toxin concentrations in San Francisco Bay, Ca, 2011–2015. Stations correspond to Fig. 1, data is displayed graphically in Fig. 2.

Particulate toxin Domoic acid [ng L�1] Microcystin toxins [ng L�1]

Station min max mean median n= min max mean median n=

657 bdl 88.7 3.9 0.0 44 bdl 461.6 18.5 2.9 446 bdl 32.9 1.6 0.0 44 bdl 53.3 3.7 1.1 4413 bdl 19.3 0.7 0.0 44 bdl 25.6 3.9 1.8 4418 bdl 316.7 12.9 0.0 75 bdl 51.1 3.7 0.0 7422 bdl 84.4 3.8 0.0 44 bdl 9.6 1.2 0.0 3127 bdl 44.0 1.9 0.0 69 bdl 70.7 4.6 0.2 6832 bdl 16.0 1.0 0.0 28 bdl 10.0 1.7 0.0 2834/36 bdl 90.3 2.0 0.0 72 bdl 22.0 3.8 0.2 72

Fig. 3. Dissolved toxin SPATT samples taken during USGS cruises from November 2011 to April 2016. Top panel is domoic acid concentrations, bottom panel is microcystinsconcentrations. SUI = Suisun Bay, SP = San Pablo Bay, CB = Central Bay, SOC = South-Central Bay, and SB = South Bay.

Table 2Solid Phase Adsorption Toxin Tracking (SPATT) samples in San Francisco Bay, Ca, 2011–2016. Stations correspond to Fig. 1, data is displayed graphically in Fig. 3.

SPATT Toxin Domoic acid [mg kg�1] Microcystin toxins [mg kg�1]

Segment min max mean median n= min max mean median n=

Suisun Bay bdl 990.7 115.8 79.8 44 bdl 25.5 2.8 0.5 44San Pablo Bay bdl 963.6 139.2 81.3 44 bdl 20.1 2.1 0.3 43Central Bay bdl 862.5 133.0 83.7 41 bdl 18.0 2.5 0.6 41South-Central Bay 4.7 1948.0 236.2 97.1 41 bdl 13.0 1.5 0.5 41South Bay 5.5 1650.5 174.0 62.5 41 bdl 23.0 1.9 0.4 41

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62.1 mg kg�1, and the Bodega Head samples had 16.7 mg kg�1

(2012) and 146 mg kg�1 (2014). Nearly half (47%) of the musselsamples collected after placement in SFB had detectable levels ofall four toxins (Fig. 4).

As previously reported in Gibble et al. (2016), microcystins weredetected in mussels sampled from April to October 2015 for 56% ofindividual mussels (n = 223). Here are the described results for thesubset of all mussels tested for multiple toxins. For that subset, 61%had detectable microcystins (n = 81), ranging from <MDL to416 mg kg�1. Domoic acid was detected in 98%, or all but two, of the

individual mussels (n = 81). The concentration of domoic acid wasvery low and ranged from <MDL to 107 mg kg�1. The PSTs weredetected in 59% of mussels (n = 73), ranging from <MDL to29.4 mg kg�1. The DSTs were detected in 71% of mussels (n = 34)ranging from <MDL to 430 mg kg�1 (Fig. 5).

4. Discussion

This study is the first to report both freshwater and marinetoxins in the same environmental mussel samples, and is the first

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Fig. 4. Toxin concentrations in mussel homogenates at each station, from left to right: domoic acid, microcystins, PSTs, and DSTs. Samples were collected from Bodega Head,CA, the open coast, just north of SFB (T-0) in May and placed at mooring locations throughout the Bay in 2012 (top panel) and 2014 (bottom panel) for June–September. Samplelocations are indicated on Fig. 1. Gray bars indicate no samples. Regulatory limits are as follows: 20,000 mg kg�1 (20 ppm) for domoic acid, 10 mg kg�1 (10 ng g�1) formicrocystins, 800 mg kg�1 (80 mg per 100 kg) for PSTs, and 160 mg kg�1 (0.16 ppm) for DSTs.

Fig. 5. Toxin concentrations in mussels for (circle) domoic acid, (triangle) microcystins, (inverted triangle) PSTs, and (square) DSTs. All samples were collected from April–September 2015 from within Central Bay. Locations are indicated on graphs and can be seen in Fig. 1. Each toxin is represented by its own y-axis. * Indicates a sample that wasabove the regulatory limit. Regulatory limits are as follows: 20,000 mg kg�1 (20 ppm) for domoic acid, 10 mg kg�1 (10 ng g�1) for microcystins, 800 mg kg�1 (80 mg per 100 kg)for PSTs, and 160 mg kg�1 (0.16 ppm) for DSTs.

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report providing evidence for accumulation and exposure to fourdistinct classes of HAB toxins in the marine food web. Co-occurrence of HAB toxins in marine shellfish has been described(MacKenzie et al., 2002) and often involves DSTs and otherlipophilic toxins including pectenotoxins and yessotoxins. Domoicacid and PSTs are strongly polar, hydrophilic toxins, and are notcommonly associated with DSP events (Dominguez et al., 2010). In2008 though, during a dolphin stranding event in Texas, USA, there

was documented co-occurrence of three HAB toxins: domoic acid,DSTs, and brevetoxins (Fire et al., 2011), where toxins found infeces and gastric contents were associated with the presence ofknown HAB species (Fire et al., 2011; Swanson et al., 2010). Thisunique combination of sampling modalities, including particulatesamples, SPATT, and accumulation by mussels provides insight intochronic toxin exposure that would not be readily apparent withsingle methods. This could be augmented with information about

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bloom toxicity, duration, and depuration rates for each of the fourtoxins, but we lack information at suitably high concurrenttemporal resolution to document exposure of the mussels.Application of depuration models or detoxification rates is alsodifficult given the lack of information about depuration of (e.g.)microcystins in mussels (Gibble et al., 2016) as well as the lack ofdata on exposure time and concentration. Notably, no correlationwas found between the presence of HAB species and toxins,suggesting that traditional phytoplankton monitoring is not anadequate substitute for toxin testing.

In 2015 there was an unprecedented toxic P. australis bloomalong the west coast of North America (McCabe et al., 2016;McKibben et al., 2017; Ryan et al., 2017). This HAB was associatedwith the Pacific Warm Anomaly (Bond et al., 2015; Di Lorenzo andMantua, 2016), and was responsible for the suspected domoic acidtoxicosis of at least 229 sea lions (2014–2015). Considering that SFBis an estuary closely coupled with the open coast Pacific Ocean(Raimonet and Cloern, 2017), there was an expectation thatevidence of the domoic acid event would be apparent within SFB,despite reported fast depuration rates for domoic acid (Blancoet al., 2002; Novaczek et al., 1992). The bloom was not apparentbased on particulate grab samples (Fig. 2) or cell counts, but isrepresented as dissolved toxin in SPATT. While there is no 1:1conversion of SPATT toxin concentration to toxin in mussels, theregulatory limit of 20,000 mg kg�1 (20 ppm; Wekell et al., 2004) isroughly equivalent to 150 mg kg�1 of SPATT resin (Lane et al., 2010).In May 2015, the peak of the open coast event (McCabe et al., 2016),the highest dissolved domoic acid concentrations throughout theBay were observed with concentrations between 618–990 mg kg�1,suggesting that there was increased dissolved domoic acid andpotential for trophic transfer of domoic acid within SFB (Fig. 3). Atthe same time, mussels collected from Central Bay where coastalexchange is expected to be influential (Raimonet and Cloern, 2017)were considerably less toxic than those collected along the opencoast (McCabe et al., 2016; Fig. 5). Only 120 km south in Santa Cruz,CA, mussel concentrations of >75,000 mg kg�1 in May and June2015 (McCabe et al., 2016) clearly documented an acute toxicitythat was not present in SFB mussels. Yet, even with the very lowconcentrations of domoic acid, all sites had elevated concen-trations in May and June when domoic acid peaked in SFB, whichfollows the temporal pattern of the bloom event along the opencoast. Thus, the temporal sequence of increasing domoic acid inspring and summer 2015 is consistent with the open coast bloom,but something inhibited toxin accumulation within SFB. Onepossibility is that the increased domoic acid seen in SFB was theresult of advection from the coast, and that temperatures withinSFB were not conducive to growth of P. australis. McCabe et al.(2016) documented maximum growth rates of P. australis isolatesfrom Monterey Bay, CA at �16 �C, followed by a decline at highertemperatures. The SFB exhibited mean temperatures of �16–20 �Cfor the 2015 water year with peak (record-breaking) temperaturesin Central Bay of >20 �C in August 2015 (Work et al., 2017). Whileunproven, the authors of this study hypothesize that domoic acidwas transported into SFB primarily as dissolved toxin (captured bySPATT), with more limited transport of intact cells (captured bymussels) and likely with temperature-based suppression ofgrowth within the SFB during that summer.

In 2015 during that same event, 29% of mussels sampled werepositive for all four toxins, while 98% had at least one detectabletoxin. Both domoic acid and PSTs, which California regulates alongthe open coast, were well below acute regulatory guidelines forquarantine of marine mussels, but more than half were contami-nated with both domoic acid and PSTs, and there were 23occurrences of acute toxin levels (Fig. 5). In 2012 and 2014 100% ofmussel homogenates were contaminated with domoic acid, 88%had microcystins, 59% had PSTs, and 94% had DSTs. Only one

sample had acute toxin levels (for microcystins; Fig. 4). Theconcentration of domoic acid and PSTs found in the musselscollected for this study would be categorized as low-level;however, the risk of long-term health effects from consumptionof these contaminated mussels is unknown. Chronic levels ofdomoic acid have been linked to a multitude of deleterious effectsin marine organisms, including persistent seizures and toxicitysyndrome in sea lions (Ferriss et al., 2017; Goldstein et al., 2008;Gulland et al., 2002), increased toxin susceptibility in zebrafish(Lefebvre et al., 2012), and a possible link between chronic domoicacid exposure and memory loss for people regularly exposed totoxin through shellfish consumption (Ferriss et al., 2017; Grattanet al., 2016). Harmful effects from chronic PSTs are similar, withreports of juvenile shellfish mortality (MacQuarrie and Bricelj,2008), morphological abnormalities in larval fish (Lefebvre et al.,2004; Silva de Assis et al., 2013), and reduced diving capabilities inmarine mammals (Durbin et al., 2002).

There is no regulatory limit for chronic exposure to domoic acidor PSTs (Adams et al., 2016; Ferriss et al., 2017), or any marinephycotoxin, and effects from chronic exposure to humans andmarine mammals are difficult to ascertain (Visciano et al., 2016).The US regulatory limit for acute exposure is based on the amountof toxin that would reasonably be ingested with a safety factor of10 for PSP and 12 for ASP assuming typical seafood consumptionrates of 100–200 g per serving. A more typical shellfish consump-tion rate is as high as 500–1000 g per serving (Wekell et al., 2004).Existing guidelines do not directly account for sensitive groups,such as pregnant women, children, recreational harvesters, orNative Americans who collect shellfish for subsistence as well asrecreational and commercial purposes, and are likely ingestingeven higher levels through repeated consumption (Adams et al.,2016; Ferriss et al., 2017; Grattan et al., 2016). As such, while thelow levels of domoic acid and PSTs found in these shellfish will notproduce acute toxicosis, the consequences of sub-lethal chronicexposure for both humans and wildlife are poorly documented.

In contrast to domoic acid and PSTs, the concentrations ofmicrocystins and DSTs were variable and on occasion considerablyhigher than regulatory guidelines of 160 mg DSTs per kg shellfish(160 mg kg�1; Lewitus et al., 2012), and 10 ng microcystins pergram of fish (10 mg kg�1; Office of Environmental Health andHazard Assessment; OEHHA); there is currently no United States orCalifornia regulatory guidelines addressing microcystin ingestionthrough shellfish consumption so it is assumed that the sameregulatory guidance can be applied to shellfish consumption.Human symptoms related to DSP are minor compared to the othertoxins discussed herein, but do include mild to moderategastrointestinal symptoms, and can lead to illness or deaththrough dehydration. These symptoms are often not reported ormisdiagnosed as pathogen induced gastroenteritis (Lewitus et al.,2012). California does not routinely monitor for DSP, though BajaCalifornia, Mexico, and the Pacific Northwest do (Lewitus et al.,2012; Trainer et al., 2013). Of the 34 mussels tested for DSTs in2015, 15% were above the regulatory guidelines, while 59% of themussels had detectable DSTs (Fig. 5). In 2014 the musselhomogenate collected from Bodega Head on the open coast(146 mg kg�1) was just below the regulatory guidelines, and in both2012 and 2014 there were low toxin levels in 94% of SFB samples.These percentages include mussels transplanted into both thenorthern Estuary and South Bay (Figs. 1 and 4); while there are nocommercial shellfisheries within SFB, recreational and subsistenceharvesters regularly collect shellfish in SFB.

As described by Gibble et al. (2016), microcystins were found inlocally occurring mussel samples from all sites, during all monthsin 2015, except August at Romberg-Tiburon Center, the mostoceanic site (Figs. 1 and 5). There was no expectation that apredominantly freshwater toxin would be found in mussels placed

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in high-salinity regions of SFB. There are precedents for freshwatercyanotoxins within an estuary (Gibble et al., 2016; Lehman et al.,2010; Paerl and Huisman, 2009; Preece et al., 2017), but theconditions during this study were not the “expected” conditionsfor high concentrations of cyanotoxins within an estuarineenvironment (Lehman et al., 2013, 2008). There was minimalfreshwater seasonal input, and California had been experiencing anunprecedented five-year drought at the time of sampling (Lehmanet al., 2017). A full 25% of the individual mussels tested exceededthe recommended regulatory guidance, though there was largevariability even among the three mussels tested per site (Fig. 5),and in the larger sample sizes reported in Gibble et al. (2016). Themussels weighed on average 5.76 � 3.5 g each. This translates toabout 17–34 mussels per person consumption, which accuratelyreflects subsistence and recreational harvester mussel consump-tion to reach the assumed ingestion of 100–200 g of shellfish usedfor regulatory purposes. These data indicate that eating just one ofthe contaminated mussels would exceed the weekly suggestedintake of microcystins, and a single meal of mussels could exceedthe suggested intake by �50x. As is common with most biotoxins,visual inspection of the mussels does not indicate contamination,and the wide range of microcystin concentrations in the musselsprovides little strategy for mitigation of exposure to the musselsabove regulatory limits. For testing purposes, homogenization ofmultiple mussels would remove some of this individual variability(Gibble et al., 2016), yet offers no additional safety for recreationalor subsistence harvesting. In 2014 microcystins were found in all ofthe homogenized mussel samples, and in all but two samples in2012 (Fig. 4). Lehman et al. (2017) describes the 2014 SFBMicrocystis bloom as the largest recorded biomass since 1999,leading to an expanded bloom. While the samples in 2014 wereless toxic than samples from 2015, there was a clear pattern ofwidespread contamination throughout SFB. Consequently, accu-mulation of this freshwater cyanotoxin in bivalves is cause foralarm in marine environments receiving freshwater runoff.

During this study, the particulate grab samples for microcystinsnever exceeded regulatory limits (Fig. 2). Regulatory guidelinesvary widely, but recent guidance from OEHHA recommend cautionwhen recreational users are exposed to 0.8 mg L�1 microcystins,while WHO identifies low risk at <10 mg L�1; the highest valueswere therefore �2 to 20-fold below those guidance levels. The grabsamples may be underrepresenting the total amount of micro-cystins in the water, since samples were collected from 1 to 2 mdepth and Microcystis cells typically float at the surface (Mloukaet al., 2004). This is consistent with the absence of Microcystis fromcell count data, which also collected at 1–2 m depth. Gibble et al.(2016) documented accumulation of dissolved toxins in mussels,highlighting the potential importance of both dissolved andparticulate phases, and the SPATT samplers indicate there ischronic, system-wide dissolved microcystins exposure to the foodweb year-round (Fig. 3).

While toxin production is often suppressed during periods ofdrought (Gibble and Kudela, 2014; Miller et al., 2010; Paerl andHuisman, 2008), there is some indication that drought conditionsin SFB can spread Microcystis blooms westward and downstream(Gibble et al., 2016; Lehman et al., 2017, 2013). This may explain thepervasiveness of the dissolved toxin found during this sampleperiod, and the high microcystin concentrations in 2014 and 2015mussel samples from the marine influenced Central and SouthBays (Figs. 4 and 5). But not to be discounted are point-source andurban run-off or smaller freshwater inputs which could beadditional, and unmeasured, sources. The bioaccumulation ofmicrocystins in marine mussels suggests the potential for acute aswell as chronic risk of toxicosis for marine mammals, marine andestuarine birds (Gibble et al., 2017), and humans, given thatconcentrations recorded from this study exceeded levels

associated with a 2009 acute marine otter mortality event inMonterey Bay, CA (Miller et al., 2010). Currently, however, there isno regulatory monitoring of the marine food web for freshwatertoxins in California.

The concentrations of algal toxins detected in SFB for domoic acidand PSTs were low relative to concentrations associated with acutemortality in marine mammals and humans (Lefebvre and Robertson,2010; Trainer et al., 2007). The concentrations for DSTs andmicrocystins were more concerning, as they frequently exceededthe regulatory limits for human consumption (Díaz et al., 2016;Preece et al., 2017). The impact of both chronic and multiple toxinexposure to marine wildlife or humans is unknown (Adams et al.,2016; Capper et al., 2013; Ferriss et al., 2017; Fire et al., 2011). In somecases, studies suggest no or unknown synergistic effects, such as forsimultaneous exposure to domoic acid and brevetoxin in bottlenosedolphins (Fire et al., 2011) or brevetoxins, okadaic acid, and saxitoxinin manatees and turtles (Capper et al., 2013). Yet, there is likely areduction in marine mammal fitness through immunosuppression(Capper et al., 2013). Further study is clearly needed to assess theconsequences of multiple, simultaneous toxin exposure in bothwildlife and humans.

5. Conclusion

San Francisco Bay exhibits simultaneous presence and bio-accumulation of at least four HAB toxin groups. Domoic acid andmicrocystins are persistent in both time and space within theestuary, while microcystins and DSTs routinely exceed regulatoryaction levels. The synergistic or additive effects for higher trophiclevel exposure to these multiple toxin suites are unknown, and areunlikely to be detected given that there has historically been noroutine regulatory monitoring within SFB for these toxins, DSTs arenot regularly monitored in California shellfish, and there is norequirement for monitoring freshwater algal toxins in marinebivalves. As with other estuaries (Preece et al., 2017), SFB may beacting as a mixing bowl and bioreactor for harmful algal speciesand toxins endemic to freshwater, brackish, and marine ecosys-tems. To the extent that SFB is representative of other estuaries,this strongly suggests that the community needs to reassessmonitoring and management practices with regard to harmfulalgae and their toxins at the land-sea interface.

Acknowledgements

Funding for this project was provided by NOAA MERHAB [grantA15NOS4780205], SFEI Contract 1084, 1006, and 1220, and NOAAIOOS [grant NA14NOS0120148]. Thank you for field support fromApplied Marine Sciences, C. Martin, T. Schraga, E. Novick, E. Nejad,R. Romero, Z. Sylvester, and the captain and crew of the R/V Polaris.Thank you for laboratory support from K. Negrey, T. Schraga, T.Fredrickson, and Z. Sylvester. This is NOAA MERHAB publicationMER209. [SS]

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